Stage-specific proteomic expression patterns of the human filarial parasite Brugia malayi and its endosymbiont Wolbachia.

Global proteomic analyses of pathogens have thus far been limited to unicellular organisms (e.g., protozoa and bacteria). Proteomic analyses of most eukaryotic pathogens (e.g., helminths) have been restricted to specific organs, specific stages, or secretomes. We report here a large-scale proteomic characterization of almost all the major mammalian stages of Brugia malayi, a causative agent of lymphatic filariasis, resulting in the identification of more than 62% of the products predicted from the Bm draft genome. The analysis also yielded much of the proteome of Wolbachia, the obligate endosymbiont of Bm that also expressed proteins in a stage-specific manner. Of the 11,610 predicted Bm gene products, 7,103 were definitively identified from adult male, adult female, blood-borne and uterine microfilariae, and infective L3 larvae. Among the 4,956 gene products (42.5%) inferred from the genome as "hypothetical," the present study was able to confirm 2,336 (47.1%) as bona fide proteins. Analysis of protein families and domains coupled with stage-specific expression highlight the important pathways that benefit the parasite during its development in the host. Gene set enrichment analysis identified extracellular matrix proteins and those with immunologic effects as enriched in the microfilarial and L3 stages. Parasite sex- and stage-specific protein expression identified those pathways related to parasite differentiation and demonstrates stage-specific expression by the Bm endosymbiont Wolbachia as well.

An insight into the sialome of the oriental rat flea, Xenopsylla cheopis (Rots).

BACKGROUND: The salivary glands of hematophagous animals contain a complex cocktail that interferes with the host hemostasis and inflammation pathways, thus increasing feeding success. Fleas represent a relatively recent group of insects that evolved hematophagy independently of other insect orders. RESULTS: Analysis of the salivary transcriptome of the flea Xenopsylla cheopis, the vector of human plague, indicates that gene duplication events have led to a large expansion of a family of acidic phosphatases that are probably inactive, and to the expansion of the FS family of peptides that are unique to fleas. Several other unique polypeptides were also uncovered. Additionally, an apyrase-coding transcript of the CD39 family appears as the candidate for the salivary nucleotide hydrolysing activity in X.cheopis, the first time this family of proteins is found in any arthropod salivary transcriptome. CONCLUSION: Analysis of the salivary transcriptome of the flea X. cheopis revealed the unique pathways taken in the evolution of the salivary cocktail of fleas. Gene duplication events appear as an important driving force in the creation of salivary cocktails of blood feeding arthropods, as was observed with ticks and mosquitoes. Only five other flea salivary sequences exist at this time at NCBI, all from the cat flea C. felis. This work accordingly represents the only relatively extensive sialome description of any flea species. Sialotranscriptomes of additional flea genera will reveal the extent that these novel polypeptide families are common throughout the Siphonaptera.

Proteomic investigation of natural killer cell microsomes using gas-phase fractionation by mass spectrometry.

We have explored the utility of gas-phase fractionation by mass spectrometry (MS) in the mass-to-charge (m/z) dimension (GPF(m/z)) for increasing the effective number of protein identifications in cases where sample quantity limits the use of multi-dimensional chromatographic fractionation. A peptide digestate from proteins isolated from the membrane fraction of natural killer (NK) cells was analyzed by microcapillary reversed-phase liquid chromatography coupled online to an ion-trap (IT) mass spectrometer. Performing GPF(m/z) using eight narrow precursor ion scan m/z ranges enabled the identification of 340 NK cell proteins from 12 microg of digestate, representing more than a fivefold increase in the number of proteins identified as compared to the same experiment employing a standard precursor ion survey scan m/z range (i.e., m/z 400-2000). The results show that GPF(m/z) represents an effective technique for increasing protein identifications in global proteomic investigations especially when sample quantity is limited.

Liquid Tissue: proteomic profiling of formalin-fixed tissues.

Identification and quantitation of candidate biomarker proteins in large numbers of individual tissues is required to validate specific proteins, or panels of proteins, for clinical use as diagnostic, prognostic, toxicological, or therapeutic markers. Mass spectrometry (MS) provides an exciting analytical methodology for this purpose. Liquid Tissue MS protein preparation allows researchers to utilize the vast, already existing, collections offormalin-fixed paraffin-embedded (FFPE) tissues for the procurement of peptides and the analysis across a variety of MS platforms.

Quantitative analysis of the low molecular weight serum proteome using 18O stable isotope labeling in a lung tumor xenograft mouse model.

With advancements in the analytical technologies and methodologies in proteomics, there is great interest in biomarker discovery in biofluids such as serum and plasma. Current hypotheses suggest that the low molecular weight (LMW) serum proteome possesses an archive of clipped and cleaved protein fragments that may provide insight into disease development. Though these biofluids represent attractive samples from which new and more accurate disease biomarkers may be found, the intrinsic person-to-person variability in these samples complicates their discovery. Mice are one of the most extensively used animal models for studying human disease because they represent a highly controllable experimental model system. In this study, the LMW serum proteome was compared between xenografted tumor-bearing mice and control mice by differential labeling utilizing trypsin-mediated incorporation of the stable isotope of oxygen, 18O. The digestates were combined, fractionated by strong cation exchange chromatography, and analyzed by nanoflow reversed-phase liquid chromatography coupled online with tandem mass spectrometry, resulting in the identification of 6003 proteins identified by at least a single, fully tryptic peptide. Almost 1650 proteins were identified and quantitated by two or more fully tryptic peptides. The methodology adopted in this work provides the means for future quantitative measurements in comparative animal models of disease and in human disease cohorts.

A proteomic characterization of the plasma membrane of human epidermis by high-throughput mass spectrometry.

Membrane proteins are responsible for many critical cellular functions and identifying cell surface proteins on different keratinocyte populations by proteomic approaches would improve our understanding of their biological function. The ability to characterize membrane proteins, however, has lagged behind that of soluble proteins both in terms of throughput and protein coverage. In this study, a membrane proteomic investigation of keratinocytes using a two-dimensional liquid chromatography (LC) tandem-mass spectrometry (MS/MS) approach that relies on a buffered methanol-based solubilization, and tryptic digestion of purified plasma membrane is described. A highly enriched plasma membrane fraction was prepared from newborn foreskins using sucrose gradient centrifugation, followed by a single-tube solubilization and tryptic digestion of membrane proteins. This digestate was fractionated by strong cation-exchange chromatography and analyzed using microcapillary reversed-phase LC-MS/MS. In a set of 1306 identified proteins, 866 had a gene ontology (GO) annotation for cellular component, and 496 of these annotated proteins (57.3%) were assigned as known integral membrane proteins or membrane-associated proteins. Included in the identification of a large number of aqueous insoluble integral membrane proteins were many known intercellular adhesion proteins and gap junction proteins. Furthermore, 121 proteins from cholesterol-rich plasma membrane domains (caveolar and lipid rafts) were identified.

The use of urine proteomic and metabonomic patterns for the diagnosis of interstitial cystitis and bacterial cystitis.

The advent of systems biology approaches that have stemmed from the sequencing of the human genome has led to the search for new methods to diagnose diseases. While much effort has been focused on the identification of disease-specific biomarkers, recent efforts are underway toward the use of proteomic and metabonomic patterns to indicate disease. We have developed and contrasted the use of both proteomic and metabonomic patterns in urine for the detection of interstitial cystitis (IC). The methodology relies on advanced bioinformatics to scrutinize information contained within mass spectrometry (MS) and high-resolution proton nuclear magnetic resonance (1H-NMR) spectral patterns to distinguish IC-affected from non-affected individuals as well as those suffering from bacterial cystitis (BC). We have applied a novel pattern recognition tool that employs an unsupervised system (self-organizing-type cluster mapping) as a fitness test for a supervised system (a genetic algorithm). With this approach, a training set comprised of mass spectra and 1H-NMR spectra from urine derived from either unaffected individuals or patients with IC is employed so that the most fit combination of relative, normalized intensity features defined at precise m/z or chemical shift values plotted in n-space can reliably distinguish the cohorts used in training. Using this bioinformatic approach, we were able to discriminate spectral patterns associated with IC-affected, BC-affected, and unaffected patients with a success rate of approximately 84%.

Quantitative proteomic analysis of human breast epithelial cells with differential telomere length.

Telomeres play important functional roles in cell proliferation, cell cycle regulation, and genetic stability, in which telomere length is critical. In this study, quantitative proteome comparisons for the human breast epithelial cells with short and long telomeres (184-hTERTL vs. 184-hTERTS and 90P-hTERTL vs. 90P-hTERTS), resulting from transfection of the human telomerase reverse transcriptase (hTERT) gene, were performed using cleavable isotope-coded affinity tags. More than 2000 proteins were quantified in each comparative experiment, with approximately 77% of the proteins identified in both analyses. In the cells with long telomeres, significant and consistent alterations were observed in metabolism (amino acid, nucleotide, and lipid metabolism), genetic information transmission (transcription and translation regulation, spliceosome and ribosome complexes), and cell signaling. Interestingly, the DNA excision repair pathway is enhanced, while integrin and its ligands are downregulated in the cells with long telomeres. These results may provide valuable information related to telomere functions.

Prostaglandin E2 is a major inhibitor of dendritic cell maturation and function in Ixodes scapularis saliva.

Tick saliva is thought to contain a number of molecules that prevent host immune and inflammatory responses. In this study, the effects of Ixodes scapularis saliva on cytokine production by bone marrow-derived dendritic cells (DCs) from C57BL/6 mice stimulated by TLR-2, TLR-4, and TLR-9 ligands were studied. Saliva at remarkably diluted concentrations (<1/2000) promotes a dose-dependent inhibition of IL-12 and TNF-alpha production induced by all TLR ligands used. Using a combination of fractionation techniques (microcon filtration, molecular sieving, and reversed-phase chromatography), we unambiguously identified PGE(2) as the salivary inhibitor of IL-12 and TNF-alpha production by DCs. Moreover, we have found that I. scapularis saliva (dilution 1/200; approximately 10 nM PGE(2)) marginally inhibited LPS-induced CD40, but not CD80, CD86, or MHC class II expression. In addition, saliva significantly suppressed the ability of DCs to stimulate Ag-specific CD4(+) T cell proliferation and IL-2 production. Notably, the effect of saliva on DC maturation and function was reproduced by comparable concentrations of standard PGE(2). These findings indicate that PGE(2) accounts for most inhibition of DC function observed with saliva in vitro. The role of salivary PGE(2) in vector-host interaction and host immune modulation and inflammation in vivo is also discussed. This study is the first to identify molecularly a DC inhibitor from blood-sucking arthropods.

Analysis of the extracellular matrix vesicle proteome in mineralizing osteoblasts.

Many key processes central to bone formation and homeostasis require the involvement of osteoblasts, cells responsible for accumulation and mineralization of the extracellular matrix (ECM). During this complex and only partially understood process, osteoblasts generate and secrete matrix vesicles (MVs) into the ECM to initiate mineralization. Although they are considered an important component of mineralization process, MVs still remain a mystery. To better understand their function and biogenesis, a proteomic analysis of MVs has been conducted. MVs were harvested by two sample preparation approaches and mass spectrometry was utilized for protein identification. A total of 133 proteins were identified in common from the two MV preparations, among which were previously known proteins, such as annexins and peptidases, along with many novel proteins including a variety of enzymes, osteoblast-specific factors, ion channels, and signal transduction molecules, such as 14-3-3 family members and Rab-related proteins. To compare the proteome of MV with that of the ECM we conducted a large-scale proteomic analysis of collagenase digested mineralizing osteoblast matrix. This analysis resulted in the identification of 1,327 unique proteins. A comparison of the proteins identified from the two MV preparations with the ECM analysis revealed 83 unique, non-redundant proteins identified in all three samples. This investigation represents the first systematic proteomic analysis of MVs and provides insights into both the function and origin of these important mineralization-regulating vesicles.

Probing early growth response 1 interacting proteins at the active promoter in osteoblast cells using oligoprecipitation and mass spectrometry.

Current advances in proteomics have allowed for a rapidly expanding integration of associated methodologies with more traditional molecular and biochemical approaches to the study of cell function. Recent studies on the role of inorganic phosphate have suggested this ion is a novel signaling molecule capable of altering the function of numerous cell types. Elevated inorganic phosphate generated in the extracellular microenvironment by differentiating osteoblasts has recently been determined to act through a largely uncharacterized mechanism as an important signaling molecule responsible for altering the transcription of various genes during osteoblast differentiation. The transcription factor, early growth response protein 1 (EGR1), has previously been shown to be involved in the early response of osteoblasts to inorganic phosphate. To elucidate the role of EGR1 as a potential early regulator of transcription in the inorganic phosphate response, an oligoprecipitation procedure was optimized to capture the DNA bound, transcriptionally active form of EGR1. The interacting proteins thusly captured were identified using mass spectrometry (MS). Proteins involved in transcription, RNA processing, and chromatin modification were identified by this approach. The combined oligoprecipitation-MS approach presented here is highly effective for isolating and characterizing entire transcriptional complexes in the DNA bound state and is broadly extendable to the identification of both known and unknown transcription factor protein complexes.

Identification of the SELDI ProteinChip human serum retentate by microcapillary liquid chromatography-tandem mass spectrometry.

Surface-enhanced laser desorption/ionization (SELDI) time-of-flight (TOF) mass spectrometry (MS) has been widely applied for conducting biomarker research with the goal of discovering patterns of proteins and/or peptides from biological samples that reflect disease status. Many diseases, ranging from cancers of the colon, breast, and prostate to Alzheimer's disease, have been studied through serum protein profiling using SELDI-based methods. Although the results from SELDI-based diagnostic studies have generated a great deal of excitement and skepticism alike, the basis of the molecular identities of the features that underpin the diagnostic potential of the mass spectra is still largely unexplored. A detailed investigation has been undertaken to identify the compliment of serum proteins that bind to the commonly used weak cation exchange (WCX-2) SELDI protein chip. Following incubation and washing of a standard serum sample on the WCX-2 sorbent, proteins were harvested, digested with trypsin, fractionated by strong cation exchange liquid chromatography (LC), and subsequently analyzed by microcapillary reversed-phase LC coupled online with an ion-trap mass spectrometer. This analysis resulted in the identification of 383 unique proteins in the WCX-2 serum retentate. Among the proteins identified, 50 (13%) are documented clinical biomarkers with 36 of these (72%) identified from multiple peptides.

Combined chemical and enzymatic stable isotope labeling for quantitative profiling of detergent-insoluble membrane proteins isolated using Triton X-100 and Brij-96.

Effective quantitative profiling of detergent-insoluble membrane proteins using high-throughput mass spectrometry (MS)-based proteomics would allow a better understanding of physiological and pathological processes that take place at the cell surface. To increase the coverage of proteins present in detergent-resistant membrane microdomains (DRMMs), a combination of 16O/18O and isotope coded affinity tags (ICAT) labeling was used in a comparative analysis of detergent-insoluble membrane proteins isolated from rat basophilic leukemia cells (RBL-2H3), with either Triton X-100 or Brij-96. The analysis resulted in the quantification of 738 unique proteins from Triton X-100 and Brij-96 isolated DRMMs, significantly exceeding the number of proteins quantified from either single labeling technique. Twenty-five noncysteine-containing proteins were quantified, as well as 32 cysteine-containing proteins that would have been missed if either 16O/18O or ICAT labeling had been used exclusively, which illustrate better proteome coverage and enhanced ability to quantitate. The comparative analysis revealed that proteins were more readily extracted using Triton X-100 than Brij-96; however, Triton X-100 also extracted larger quantities of non-DRMMs-associated proteins. This result confirms previous, targeted studies suggesting that DRMMs isolated using Triton X-100 and Brij-96 differ in their protein content.

Proteomic analysis of plasma membrane from hypoxia-adapted malignant melanoma.

Hypoxic conditions often persist within poorly vascularized tumors. At the cellular level constitutive activation of transcriptional regulators of the hypoxic response leads to the emergence of clones with aggressive phenotypes. The primary interface between the cell and the hypoxic environment is the plasma membrane. A detailed investigation of this organelle is expected to yield further targets for therapeutic perturbation of the response to hypoxia. In the present study, quantitative proteomic analysis of plasma membrane from hypoxia-adapted murine B16F10 melanoma was performed using differential 16O/18O stable isotopic labeling and multidimensional liquid chromatography-tandem mass spectrometry. The analysis resulted in the identification of 24,853 tryptic peptides, providing quantitative information for 2,433 proteins. For a subset of plasma membrane and secreted proteins, quantitative RT-PCR was used to gain further insight into the genomic regulatory events underlying the response to hypoxia. Consistent increases at the proteomic and transcriptomic levels were observed for aminopeptidase N (CD13), carbonic anhydrase IX, potassium-transporting ATPase, matrix metalloproteinase 9, and stromal cell derived factor I (SDF-1). Antibody-based analysis of a panel of human melanoma cell lines confirmed that CD13 and SDF-1 were consistently upregulated during hypoxia. This study provides the basis for the discovery of novel hypoxia-induced membrane proteins.

Parallel analysis of transcript and translation profiles: identification of metastasis-related signal pathways differentially regulated by drug and genetic modifications.

Tumor metastasis is a complex multistep process normally involving dysregulation of multiple signal transduction pathways. In this study, we developed a novel approach to efficiently define dysreguated pathways associated with metastasis by comparing global gene and protein expressions of two distinct metastasis-suppressed models. Consequently, we identified common features shared by the two models which are potentially associated with metastasis. The efficiency of metastasis from the highly aggressive polyoma middle T-induced mouse mammary tumors was suppressed by either prolonged caffeine exposure or by breeding the animal to a low metastatic mouse strain. Molecular profiles of the primary tumors from both metastasis-suppressed classes were then derived to identify molecules and pathways that might underlie a common mechanism of metastasis. A number of differentially regulated genes and proteins were identified, including genes encoding basement membrane components, which were inversely related to metastatic efficiency. In addition, the analysis revealed that the Stat signal transduction pathways were potentially associated with metastasis inhibition, as demonstrated by enhanced Stat1 activation, and decreased Stat5 phosphorylation in both genetic and pharmacological modification models. Tumor cells of low-metastatic genotypes also demonstrated anti-apoptotic properties. The common changes of these pathways in all of the metastasis-suppressed systems suggest that they may be critical components in the metastatic cascade, at least in this model system. Our data demonstrate that analysis of common changes in genes and proteins in a metastatic-related context greatly decrease the complexity of data analysis, and may serve as a screening tool to identify biological important factors from large scale data.

Mass spectrometry reveals specific and global molecular transformations during viral infection.

Mass spectrometry analysis was used to target three different aspects of the viral infection process: the expression kinetics of viral proteins, changes in the expression levels of cellular proteins, and the changes in cellular metabolites in response to viral infection. The combination of these methods represents a new, more comprehensive approach to the study of viral infection revealing the complexity of these events within the infected cell. The proteins associated with measles virus (MV) infection of human HeLa cells were measured using a label-free approach. On the other hand, the regulation of cellular and Flock House Virus (FHV) proteins in response to FHV infection of Drosophila cells was monitored using stable isotope labeling. Three complementary techniques were used to monitor changes in viral protein expression in the cell and host protein expression. A total of 1500 host proteins was identified and quantified, of which over 200 proteins were either up- or down-regulated in response to viral infection, such as the up-regulation of the Drosophila apoptotic croquemort protein, and the down-regulation of proteins that inhibited cell death. These analyses also demonstrated the up-regulation of viral proteins functioning in replication, inhibition of RNA interference, viral assembly, and RNA encapsidation. Over 1000 unique metabolites were also observed with significant changes in over 30, such as the down-regulated cellular phospholipids possibly reflecting the initial events in cell death and viral release. Overall, the cellular transformation that occurs upon viral infection is a process involving hundreds of proteins and metabolites, many of which are structurally and functionally uncharacterized.

Proteomic analysis of lipid microdomains from lipopolysaccharide-activated human endothelial cells.

The endothelium plays a critical role in orchestrating the inflammatory response seen during sepsis. Many of the inflammatory effects of Gram-negative sepsis are elicited by lipopolysaccharide (LPS), a glycolipid component of bacterial cell walls. Lipid-rich microdomains have been shown to concentrate components of the LPS signaling system. However, much remains to be learned about which proteins are constituents of lipid microdomains, and how these are regulated following cell activation. Progress in this area would be accelerated by employing global proteomic analyses, but the hydrophobicity of membrane proteins presents an analytical barrier to the effective application of such approaches. Herein, we describe a method to isolate detergent-resistant membranes from endothelial cells, and prepare these samples for proteomic analysis in a way that is compatible with subsequent separations and mass spectrometric (MS) analysis. In the application of these sample preparation and MS analyses, 358 proteins from the lipid-rich microdomains of LPS-activated endothelial cell membranes have been identified of which half are classified as membrane proteins by Gene Ontology. We also demonstrate that the sample preparation method used for solubilization and trypsin digestion of lipid-rich microdomains renders the membrane spanning sequences of transmembrane proteins accessible for endoproteolytic hydrolysis. This analysis sets the analytical foundation for an in-depth probing of LPS signaling in endothelial cells.

Quantitative proteomic analysis of sokotrasterol sulfate-stimulated primary human endothelial cells.

The endothelium forms a continuous monolayer at the interface between blood and tissue and contributes significantly to the sensing and transducing of signals between blood and tissue. New blood vessel formation, or angiogenesis, is initiated by the activation of endothelial cells and is an important process required for various pathological and physiological situations. This study used cleavable isotope-coded affinity tag reagents combined with mass spectrometry to investigate the molecular basis of a recently discovered angiogenesis-promoting steroid, sokotrasterol sulfate. Changes in the relative abundances of over 1000 proteins within human endothelial cells treated with sokotrasterol sulfate and vehicle-treated cells were identified and quantitated using this technique. A method that examines the entire ensemble of quantitative measurements was developed to identify proteins that showed a statistically significant change in relative abundance resulting from treatment with sokotrasterol sulfate. A total of 93 proteins was significantly up-regulated, and 37 were down-regulated in response to sokotrasterol sulfate stimulation of endothelial cells. Among the up-regulated proteins, several were identified that are novel to endothelial cells and are likely involved in cell communication and morphogenesis. These findings are consistent with a role for sokotrasterol sulfate in endothelial sprouting.

Investigation of the mouse serum proteome.

With the rapid assimilation of genomic information and the equally impressive developments in the field of proteomics, there is an unprecedented interest in biomarker discovery. Although human biofluids represent increasingly attractive samples from which new and more accurate disease biomarkers may be found, the intrinsic person-to-person variability in these samples complicates their discovery. One of the most extensively used animal models for studying human disease is mouse because, unlike humans, they represent a highly controllable experimental model system. Unfortunately, very little is known about the proteomic composition of mouse serum. In this study, a multidimensional fractionation approach on both the protein and the peptide level that does not require depletion of highly abundant serum proteins was combined with tandem mass spectrometry to characterize proteins within mouse serum. Over 12 300 unique peptides that originate from 4567 unique proteins-approximately 16% of all known mouse proteins-were identified. The results presented here represent the broadest proteome coverage in mouse serum and provide a foundation from which quantitative comparisons can be made in this important animal model.

A combined proteome and microarray investigation of inorganic phosphate-induced pre-osteoblast cells.

Inorganic phosphate, which is generated during osteoblast differentiation and mineralization, has recently been identified as an important signaling molecule capable of altering signal transduction pathways and gene expression. A large scale quantitative proteomic investigation of pre-osteoblasts stimulated with inorganic phosphate for 24 h resulted in the identification of 2501 proteins, of which 410 (16%) had an altered abundance ratio of greater than or equal to 1.75-fold, either up or down, revealing both novel and previously defined osteoblast-regulated proteins. A pathway/function analysis of these proteins revealed an increase in cell cycle and proliferation that was subsequently verified by conventional biochemical means. To further analyze the mechanisms by which inorganic phosphate regulates cellular protein levels, we undertook a mRNA microarray analysis of pre-osteoblast cells at 18, 21, and 24 h after inorganic phosphate exposure. Comparison of the mRNA microarray data with the 24-hour quantitative proteomic data resulted in a generally weak overall correlation; the 21-hour RNA sample showed the highest correlation to the proteomic data. However, an analysis of osteoblast relevant proteins revealed a much higher correlation at all time points. A comparison of the microarray and proteomic datasets allowed for the identification of a number of candidate proteins that are post-transcriptionally regulated by elevated inorganic phosphate, including Fra-1, a member of the activator protein-1 family of transcription factors. The analysis of the data presented here not only sheds new light on the important roles of inorganic phosphate in osteoblast function but also begins to address the contribution of post-transcriptional and post-translational regulation to a cell's expressed proteome. The ability to accurately measure changes in both protein abundance and mRNA levels on a system-wide scale represents a novel means to extract data from previously one-dimensional datasets.